The Saccharomyces cerevisiae Sln1 protein is a 'twocomponent' regulator involved in osmotolerance. Twocomponent regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.

Ras oncogene proteins are plasma membrane-associated signal transducers that are found in all eukaryotes. Posttranslational addition of lipid to a carboxyl-terminal CaaX box (where "C" represents a cysteine, "a" is generally an aliphatic residue, and X can be any amino acid) is required to target Ras proteins to the cytosolic surface of the plasma membrane. The pathway by which Ras translocates from the endoplasmic reticulum to the plasma membrane is currently not clear. We have performed a genetic screen to identify components of the Ras plasma membrane localization pathway. Mutations in two genes, ERF2 and ERF4/SHR5, have been shown to affect the palmitoylation and subcellular localization of Ras proteins. In this report, we show that Erf4p is localized on the endoplasmic reticulum as a peripheral membrane protein in a complex with Erf2p, an integral membrane protein that was identified from the same genetic screen. Erf2p has been shown to be required for the plasma membrane localization of GFP-Ras2p via a pathway distinct from the classical secretory pathway (X. Dong and R. J. Deschenes, manuscript in preparation). We show here that Erf4p, like Erf2p, is involved in the plasma membrane localization of Ras2p. Erf2p and Erf4p represent components of a previously uncharacterized subcellular transport pathway involved in the plasma membrane targeting of Ras proteins.Ras proteins are plasma membrane-bound small GTPases that regulate signal transduction pathways by cycling between GTP-and GDP-bound forms (1, 2). Ras proteins are initially synthesized as cytosolic precursors, but then undergo modifications at a carboxyl-terminal motif called the CaaX box (where "C" represents a cysteine, "a" is generally an aliphatic residue, and "X" can be any amino acid) (3). These modifications include farnesylation of the CaaX box cysteine, proteolysis of the -aaX, and carboxyl methylation (4 -8). The last two steps occur on the cytosolic surface of the ER. 1 Most Ras proteins, including yeast Ras1p and Ras2p and mammalian H-Ras and N-Ras, are further modified by palmitoylation on one or two additional cysteine residues often found adjacent to the CaaX box. Not all prenylated Ras proteins undergo palmitoylation. Mammalian K-Ras4B, for example, lacks a palmitoylation site but contains multiple basic residues near the C terminus that are required for plasma membrane targeting (4, 9 -11). These observations have led to a two-signal hypothesis for trafficking in which CaaX box processing plus at least one additional signal is required for plasma membrane localization of Ras (12).The mechanism by which Ras and other prenylated proteins are transported from the cytoplasmic surface of the ER to the plasma membrane is not clear. The classical secretory pathway, which has been explored extensively by genetic studies in S. cerevisiae and biochemical fractionation of mammalian cell lines, is an obvious candidate (13-15). Many proteins are transported via the classical secretory pathway by a process of vesicle budding and fusion (16,17). Li...

Summary The yeast histidine kinase, Sln1p, is a plasma membrane‐associated osmosensor that regulates the activity of the osmotic stress MAP kinase pathway. Changes in the osmotic environment of the cell influence the autokinase activity of the cytoplasmic kinase domain of Sln1p. Neither the nature of the stimulus, the mechanism by which the osmotic signal is transduced nor the manner in which the kinase is regulated is currently clear. We have identified several mutations located in the linker region of the Sln1 kinase (just upstream of the kinase domain) that cause hyperactivity of the Sln1 kinase. This region of histidine kinases is largely uncharacterized, but its location between the transmembrane domains and the cytoplasmic kinase domain suggests that it may have a potential role in signal transduction. In this study, we have investigated the Sln1 linker region in order to understand its function in signal transduction and regulation of Sln1 kinase activity. Our results indicate that the linker region forms a coiled‐coil structure and suggest a mechanism by which alterations induced by osmotic stress influence kinase activity by altering the alignment of the phospho‐accepting histidine with respect to the catalytic domain of the kinase.

In this article I present some parts of the "story" of MSG that might be of most interest to chemists, chemistry teachers, and their students. HistoryGlutamic acid was first isolated as a pure substance in 1866 by the German chemist Ritthausen through the acidic hydrolysis of gliadin, a component of wheat gluten. It was not until 1908, however, that the Japanese chemist Kikunae Ikeda found that glutamic acid was responsible for the flavor-enhancing properties of the kelplike seaweed, "konbu", or Laminaria Japonica, that had been used for many centuries in Japan in the preparation of soup stocks. By extracting 40 kilograms of the seaweed with hot water, Ikeda obtained 30 grams of (S )-glutamic acid, which he then identified as the taste-enhancing component of konbu. Ikeda immediately patented a process for isolating monosodium glutamate from wheat flour, and in 1909 the first monosodium glutamate was produced commercially under the trade name Ajinomoto (Aji no moto; "at the origin of flavor").Glutamic acid has now been isolated from innumerable vegetable sources, of which the most practically useful have included wheat gluten, soybean meal, casein, and the residue from the Steffen process for the production of beet sugar, the so-called "Steffen waste". The preparation of (S )-glutamic acid from wheat gluten is described in Organic Syntheses, Collective Volume 1 (1).Since 1908 the sodium salt of glutamic acid, or MSG, has come into use around the world as an additive, or seasoning, to enhance the flavor of foods. MSG is usually used in combination with salt, and, in general, a suitable quantity of MSG is 10-20% of the quantity of salt to be added. The connection between MSG and taste is described in more detail below. Commercial Production of Amino Acids(S )-Glutamic acid, relative to other pure amino acids, is produced in the largest quantities around the world. Its two closest competitors are (S )-lysine, and (R,S )-methionine, as indicated in Table 1.

G protein-coupled receptors (GPCRs) form a class of biological chemical sensors with an enormous diversity in ligand binding and sensitivity. To explore structural aspects of ligand recognition, we subjected the human UDP-glucose receptor (P2Y14) functionally expressed in the yeast Saccharomyces to directed evolution. We sought to generate new receptor subtypes with ligand-binding properties that would be useful in the development of practical biosensors. Mutagenesis of the entire UDP-glucose receptor gene yielded receptors with increased activity but similar ligand specificities, while random mutagenesis of residues in the immediate vicinity of the ligand-binding pocket yielded mutants with altered ligand specificity. By first sensitizing the P2Y14 receptor and then redirecting ligand specificity, we were able to create mutant receptors suitable for a simple biosensor. Our results demonstrate the feasibility of altering receptor ligand-binding properties via a directed evolution strategy, using standard yeast genetic techniques. The novel receptor mutants can be used to detect chemical ligands in complex mixtures and to discriminate among chemically or stereochemically related compounds. Specifically, we demonstrate how engineered receptors can be applied in a pairwise manner to differentiate among several chemical analytes that would be indistinguishable with a single receptor. These experiments demonstrate the feasibility of a combinatorial approach to detector design based on the principles of olfaction.